【作者】 王莹；

【导师】 韩烈保；

【作者基本信息】 北京林业大学 ， 草业科学， 2008， 硕士

【摘要】 Rd29A基因启动子是一个受干旱、高盐和低温等逆境诱导表达的特异性启动子,也是植物抗逆性基因工程中理想的逆境诱导型启动子。本研究的目的是从结缕草中分离出Rd29A基因启动子,并对其序列进行测序、比较和分析,鉴定该启动子的功能。筛选出适用于草坪草转基因的诱导型启动子,促进草坪草转基因技术的发展,对于逆境条件下结缕草的生长发育有重要意义。根据文献上发表的Rd29A基因启动子序列设计并合成了特异引物,以我国野生的结缕草为实验材料,通过PCR方法从该结缕草样品基因组DNA中扩增到目的片断,并进行了测序、比较和分析,与GenBank报道的Rd29A基因启动子序列相比较,同源性为41.65%。该序列在GenBank上的登录号是:EU346948。利用GFP和GUS基因作为报告基因,构建了用于比较鉴定所克隆启动子活性pBIG(35S-GFP)、pBIRG (Rd29A-GFP)和pBI(35S-GUS)、pBIR (Rd29A-GUS)四个植物真核表达载体,采用基因枪法对洋葱表皮细胞进行遗传转化。在胁迫诱导后,检测目的片段在受体细胞中调控基因表达的活性,从而鉴定其功能。结果表明克隆到的目的片段具有启动子功能,并且在干旱、高盐、低温诱导胁迫下更能强烈地驱动下游基因的表达。更多还原

【Abstract】 The promoter Rd29A is induced by drought, high-salt and cold. It is also a ideal inducible promoter in gene engineering of plant resist contrary. Our purpose is to isolate Rd29A promoter gene fragment from Zoysia japonica Steud genomic DNA. Then measuring, comparing and analyzing the order, and indentifying the function of the promoter Rd29A. We screen out inducible promoter to suit and promote transfer-gene technology in turfgrass. The study will be very important for turfgrass growth in resist contrary.According to the reported sequence of promoter Rd29A, designe and synthesize distinctive primers. We amplify the DNA fragments of Rd29A promoter through method of PCR from Zoysia japonica Steud genomic DNA, then measuring, comparing and analyzing the order. It indicates DNA sequence have 41.65% homology with the Rd29A promoter which has been submitted to GenBank. The accession number is EU346948 in GenBank.We utilize the gene of green fluorescence protein (GFP) andβ-GUS coronidaSe (GUS) as reporting genes, four transient plant expression vectors pBIG(35S-GFP), pBIRG(Rd29A-GFP) and pBI(35S- GUS), pBIR(Rd29A-GUS) are constructed. They are transformed the epidermis cells of onion by particle bombardment. After coerce inducib, detecting purpose fragment to regulate and control gene expression in acceptor cell, identify whose function thereby.Result shows that cloning purpose fragment has the function of promoter, and it can regulate lower reaches gene expression intensely in drought, high-salt and cold.更多还原